There is strong clinical and scientific interest in finding new sources of stem cells which are readily available, since stem cells have a great importance for biological studies, stem cells therapies and regenerative medicine.
Two main types of mammalian stem cells exist: embryonic stem cells (hereinafter abbreviated “eSC”) and “adult” or “somatic” stem cells (hereinafter abbreviated “sSC”).
The former type was isolated from human embryos more than one decade ago (Thomson et al., Science, 282(5891): 1145-1147, 1998). Thomson et al. discovered a method to derive and isolate these cells from embryos and foetal germ cells. eSC have the potential to develop into almost all of the more than 200 different known human body cells.
The second type of known stem cells is undifferentiated cells that are found in differentiated somatic tissue. Generally these cells are multipotent, i.e. having the capacity to differentiate into several types of somatic cell within the tissue in which they reside.
During the last ten years, sSC have been found in many organs and tissues, including central nervous system, bone marrow, peripheral blood, blood vessels, umbilical cordon blood, skeletal muscle, epidermis of the skin, dental pulp, heart, gut, liver, pancreas, lung, adipose tissue, ovarian epithelium, retina, cornea and testis. They are thought to reside in a specific area of each tissue, which is called a “stem cell niche”.
In vivo, the main role of sSC is to maintain homeostasis and to replace cells that have died because of use, apoptosis, injury or disease. However, most sSC have a limited capacity to handle major trauma or diseases that would cause a vast loss of cells and tissue.
Both types of stem cells have the ability to proliferate while maintaining an undifferentiated state and the capacity to give rise to a succession of mature functional differentiated cells.
However, whereas eSC are pluripotent, i.e. they can differentiate into almost all cell types of the body and possess the capability of developing into any organ or tissue type, sSc are multipotent, which means that sSC can only differentiate into several types of cell which are closely related to the tissue from which sSC derive. For example, a hematopoietic stem cell may only give rise to any of the different types of terminally differentiated blood cells.
Even if the embryonic stem cells have the greatest degree of differentiation potential, they are not readily available and procurement of these cells from embryos or foetal tissue, including abortuses, raises religious and ethical issues.
On the contrary, several types of sSC such as mesenchymal stem cells, hematopoietic stem cells, skin stem cells, adipose-derived stromal stem cells, are more accessible and provoke less ethical controversy than eSC. Further, in some cases stem cells can be obtained from the patient to be engrafted, i.e. the recipient which is better suitable for transplantation purpose than eSC since autologous graft avoids the risk of rejection.
Unfortunately, sCS are rare and present in small quantity in somatic tissues. Since very few stem cells are present in adult tissues, extraction procedures to recover these cells from a tissue generally result in contamination by other cell types such as fibroblasts. Consequently, when a tissue sample is harvested, it is necessary to sort a heterogeneous mixture comprising stem cells and non-stem cells. However, a specific cellular marker characterising one type of sSC does not always exist, making it difficult to isolate sSC from other cell-types of the sample. Hence, to date, isolation of normally occurring populations of stem cells from adult tissues is technically difficult and costly.
Moreover, even if sCS are more readily available than eSC, a major issue which remains to be solved is the acute organ and bone marrow shortage mainly due to the small number of donors, and which is increased by the difficulty to find matching donors, especially for minority groups.
Accordingly, there is a need for a new source of readily available stem cells. Furthermore, there is also a great need for a simple method which enables for specifically selecting any type of stem cell from biological material comprising non-stem cells.
Recently, it has been shown that neural stem cells from rat can be isolated from the brain of deceased adult or early postnatal rats even 6 days after death when rats were stored at 4° C. (Yi Xu et al., Journal of Neuroscience Research, 74: 533-540, 2003). Then, Yi Xu et al. suggested using cadavers as a new source of neural stem cells usable for clinical purposes. Further, the study of Yi Xu et al. showed that the amount of stem cells obtained from rat cadavers depends on the age of the rats, since there are significantly more neural stem cells in the early postnatal rats than in the adults. In addition, Yi Xu et al. showed that from day 2 post-mortem the number of neural stem cells strongly decreases and that only few neural stem cells survive 4 days after death in 4 weeks old rats.
A recent study performed on human neural stem cells indicates that the highest proliferation rate is obtained when these cells are cultured in hypoxic conditions, at an oxygen concentration between 2.5 and 5%, while 1% of oxygen is detrimental for cell survival (Santilli et al., PLOS One, 5(1): e8575, 2010). This study suggests that an oxygen concentration of less that 2.5% is noxious for stem cells.
Another study conducted on human abortuses preserved at 4° C. clearly showed that the number of viable neural stem cells decreased sharply when preservation is prolonged to 12 hours after death (Xinchun Liu and al., Journal of Neuroscience methods, 157: 64-70, 2006). Viable neural progenitor cells from human cadavers were also obtained from brain tissue 20 hours after death (Palmer et al., Nature, 411: 42-43, 2001).
It was also proposed in the International Application WO 01/53462 to use tissues of cadavers as a source of progenitors and stem cells, in particular as a source of progenitor cells having the capacity to develop into hepatocytes and biliary cells. WO 01/53462 recommended to use tissues which had been harvested within about six hours after the donor's heartbeat ceased, and a maximum of 30 hours post-mortem was indicated for liver tissue. However, it is to be noted that this application showed experiments only for liver progenitor cells which come from livers obtained not later than 30 hours after death.
It was also shown that the cell viability of adult rat neural stem cells when cultured 24 hours in anoxic condition at 37° C. increased up to 60% compared to normoxic condition (Bürgers et al., Exp. Brain Res., 188(1): 33-43, 2008), and that cell division activity increased from 2% in normoxic condition to 16% in anoxic condition. However, the effect of longer periods in anoxia on viability of stem cells was not tested and the transplantation potential of stem cells was not assessed.
Concerning neural stem cells, histological and immunohistochemical assays suggest that the rich vascular bed presents in brain, in particular the subventricular zone, might be an important element responsible for survival of neural stem cells during the post-mortem period and can likely act as a niche for the maintenance of neural stem cells (Yi Xu et al., Journal of Neuroscience Research, 74: 533-540, 2003).
As indicated above, the prior art mainly concerns neural stem cells and does not demonstrate that other type of stem cells can be obtained from cadavers, except liver stem cells. Further, the longest period of time following death after which viable stem cells could be found was 6 days for rat neural stem cells. Viability of human neural stem cells was assessed only until 20 hours post mortem.
In addition, because the biological environment of neural stem cells is specific and not found in other tissues, data of the prior art relating to neural stem cells do not allow to conclude that other types of stem cells can be obtained from cadavers.